Note: Descriptions are shown in the official language in which they were submitted.
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Antibodies or fragments thereof for use in the treatment of ocular diseases.
The present invention relates to the field of medical approaches for ocular
diseases that may lead to partial or total blindness. The invention provides
useful tools to be applied in medicine, including antibodies or fragments
thereof.
BACKGROUND ART
Several are the diseases that affect the retina (retinal diseases) including
age
related macular degeneration, retinitis pigmentosa, diabetic retinopathy,
macular edema and other inherited retinal degenerations, uveitis, retinal
detachment, and eye cancers. The retina is the light sensitive portion of the
eye, and is a complex tissue containing specialized photoreceptor cells, the
cones and the rods. The photoreceptors connect to a network of nerve cells
for the local processing of visual information, which is sent to the brain for
obtaining a visual image. The rods are mostly located away from the centre of
the eye in the retinal periphery. The highest concentration of cones is found
at the center of the retina, the macula, which is necessary for visual acuity.
Under the retina is located the choroid. The retinal pigment epithelium (RPE)
is a monolayer of pigmented cells situated between the neuroretina and the
choroids. The RPE is the pigmented cell layer just outside the neurosensory
retina that nourishes retinal visual cells. RPE cells protect, support, and
feed
the light sensitive retina. The dysfunction, disruption and/or loss of these
RPE
cells play a critical role in the development of the vision loss. Thus, RPE
cells are often the first cells to degenerate or suffer damage as a result of
a
traumatizing event or condition.
Another structure of the eye being of great importance in many of the retinal
diseases is the blood-retinal barrier (BRB), also called hemato-retinal
barrier.
The BRB is constituted by the inner blood-retinal barrier and the external
blood-retinal barrier. Inner blood-retinal barrier is formed by the tight
junctions
of endothelial cells. External blood-retinal barrier is constituted by the
RPE,
which cells are also connected by tight junctions. Tight junctions between
RPE cells are essential to control the transport of liquid and soluble
compounds through the BRB, as well as to avoid any toxics into the retina.
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Therefore, RPE is a key component of the external blood-retinal barrier to
assure retina integrity. The two most frequent retinal diseases that are due
to
an impairment of the external BRB thus resulting in retinal edema are diabetic
macular edema and age-related macular degeneration. In addition, alteration
of the BRB occurs also in a wide variety of ocular situations, such as
uveitis,
trauma, intraocular surgery, vascular retinopathies, hereditary dystrophies,
etc. (Cunha-Vaz et al., "The Blood¨Retinal Barrier in Retinal Disease",
European Ophthalmic Review -2009, Vol. No. 3, pp.:105-108).
Several approaches to identify the causes of many of the retinal diseases
have been performed. One of them is based on the performance of proteomic
analysis of vitreous humour. The vitreous humour is the clear gel that fills
the
space between the lens and the retina of the eyeball of humans and other
vertebrates. It is often referred to as the vitreous body or simply "the
vitreous".
The proteomic analysis by differential gel electrophoresis of the vitreous has
been applied for the characterization of the proteome in Proliferative
Diabetic
Retinopathy and in other ocular pathologies such as Diabetic Macular
Edema. Examples of this are found in Ramirez et al., "Proteomic Analysis of
Human Vitreous Fluid by DIGE: a New Strategy for Identifying Potential
Candidates in the Pathogenesis of Proliferative Diabetic Retinopathy",
Diabetologia 2007, Vol. 50, pp.:1294-1303; in Gao et al., "Characterization of
Vitreous Proteome in Diabetes without Diabetic Retinopathy and Diabetes
with Proliferative Diabetic Retinopahty", Journal of Proteome Research -
2008, vol. 7, pp. 2516-2525; and in Hernandez et al. "New pathogenic
candidates for diabetic macular edema detected by proteomic analysis",
Diabetes Care 2010; 33:e92.
Nowadays most of the treatments of the diseases affecting the retina are
implemented in advanced stages of the diseases rather than to arrest or
prevent their development. Thus, in the particular case of macular edema or
in some retinopathies (proliferative or non-proliferative diabetic
retinopathy)
the treatment is usually based on laser photocoagulation, vitrectomy and
corticosteroids intravitreal injections. All these treatments are encouraged
in
late-stages of these diseases, that is, there are no effective early
treatments.
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Moreover, they imply many side effects (pain, inflammation, hemorrhage, etc.)
and a high ratio of failures is in addition observed.
It is worth mentioning that all these diseases are of great impact, not only
because they lead to blindness or to altered vision impeding people to
develop normal life (working, walking, driving, etc.), but also because they
are
generally linked with highly prevalent disorders, such as diabetes mellitus
(in
particular the type 2), and age-related macular degeneration (AMD). The
relation with high prevalent disorders makes in turn all these retinal
disorders
of common presence in the society, thus representing a challenge for the
health institutions.
Other therapeutic approaches are based on the injection of compounds able
to block the vascular endothelial growth factor (VEGF), such as the antibody
Ranibizumab (trade name Lucentis), which has been approved to treat the
"wet" (also known as exudative or neovascular) type of age-related macular
degeneration (AMD), a common form of age-related vision loss. The antibody
is intravitreally injected once a month. The most common side effects
associated to this treatment in clinical trials were conjunctive hemorrhage,
eye pain, vitreous floaters, increased intraocular pressure, and intraocular
inflammation.
Thus, there is a need of additional therapeutically approaches to face all the
diseases in which retina is affected, in particular those diseases wherein RPE
and BRB are compromised.
SUMMARY OF THE INVENTION
Facing with the problem of finding further therapeutic approaches to treat
diseases that affect the retina, or retinal diseases, the inventors provide a
new method based on the inhibition of a key compound involved in these
pathologies, the hemopexin.
Thus, in a first aspect the invention relates to an antibody or a fragment
thereof that specifically binds to hemopexin for use in the treatment and/or
prevention of a retinal disease in which there is a dysfunction of the
external
blood retinal barrier, being the dysfunction an alteration or impairment of
the
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blood-retinal barrier for any etiology. Thus, the antibody or fragment thereof
is
for the use in the treatment and/or prevention of a retinal disease cursing
with
dysfunction of the external blood-retinal barrier, being the dysfunction an
alteration or impairment of the blood-retinal barrier for any etiology.
The first aspect of the invention can alternatively be formulated as a method
for treating and/or preventing retinal diseases in which there is a
dysfunction
(or which is the same, curses with a dysfunction) of the external blood
retinal
barrier, being the dysfunction an alteration or impairment of the blood-
retinal
barrier for any etiology, the method comprising administering an antibody or a
fragment thereof that specifically binds to hemopexin to the subject in need
thereof. This aspect can alternatively be formulated as the use of an antibody
or a fragment thereof that specifically binds to hemopexin for the manufacture
of a medicament for the treatment and/ or prevention of a retinal disease in
which there is a dysfunction (or curses with a dysfunction) of the external
blood retinal barrier, being the dysfunction an alteration or impairment of
the
blood-retinal barrier for any etiology.
Hemopexin (HPX), also known as beta-13-glycoprotein is a protein that in
humans is encoded by the HPX gene and belongs to hemopexin family of
proteins. Human HPX gene is located at chromosome 11 and corresponds to
the GenBank entry 3263. The human translated protein has 462 amino acids
and corresponds to SEQ ID NO: 1, also identified as P02790, Version 2 of
October 1, 1996 from UniPrit/SwissProt.
Hemopexin binds heme group with the highest affinity of any known protein.
Its physiological function is scavenging the heme group released or lost by
the turnover of heme proteins such as hemoglobin, and thus it protects the
body from the oxidative damage that free heme can cause. In addition,
hemopexin releases its bound ligand for internalization upon interacting with
a specific receptor situated on the surface of liver cells. This function of
hemopexin is to preserve the body's iron. Hemopexin is basically synthesized
in the liver, but there exist evidences of synthesis in the brain and the
retina.
In the state of the art, hemopexin has been used as biomarker for acute heart
failure as indicated in WO 2008087049, as biomarker for diabetic
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nephropathy, according to KR 100792630 and WO 2008141285, and as
biomarker of therapy test in leukemia according to JP 2002220349.
Surprisingly, and as will be illustrated in the examples below, hemopexin is
5 able to disrupt the RPE. Based on this fact, there are herewith provided
antibodies or fragments thereof which can specifically bind to several
hemopexin epitopes, and can block the interaction of hemopexin with its
receptor sited in the retina. All of these antibodies prevent the disruption
of
the RPE, or allow the restitution of the disrupted RPE which is forming part
of
the outer (or external) BRB.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an immunofluorescence image showing tight junction protein ZO-1
(grey lines between cells) taken by confocal microscopy of a monolayer of
ARPE-19 cells. Nuclei (DAPI tinction; shown as mostly spherical aggregates)
are appreciated as light grey agglomerates (originally blue when visualized in
the microscopy). C means control; HPX means cell monolayer after
hemopexin treatment; HPX+Ab D17 means treatment with hemopexin and
further goat anti-human hemopexin antibody; HX+Ab H-300 means treatment
with hemopexin and further rabbit anti-human hemopexin antibody; and
HX+Ab 013 means treatment with hemopexin and further mouse anti-human
hemopexin antibody.
FIG. 2 is a bar diagram showing the dextrane permeability of ARPE-19 cells
with different external conditions. Abbreviations mean the same as in FIG. 1.
3M and 180M mean 3 minutes and 180 minutes, respectively.
DETAILED DESCRIPTION OF THE INVENTION
Following definitions are included in order to facilitate comprehension of the
invention.
In the context of the present invention, "epitope" is understood to mean the
part of a peptide type macromolecule (or of an antigen), whose sequence
and/or spatial configuration is recognized by the immune system (antibodies,
T cells, B cells)
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A "fragment of an antibody" refers to a part of the antibody which is of
enough
size and appropriate structure to bind to an epitope present in the hemopexin.
Examples of fragments include F(ab), F(ab") and Fv.
The term "retinal disease" means any disease in which the retina is affected
due to multiple and variant etiologies.
A retinal disease "cursing with dysfunction of the external blood-retinal
barrier" or "in which there is a dysfunction of the external blood-retinal
barrier", includes all retinal diseases in which the external blood-retinal
barrier is altered or impaired for any etiology (Cunha-Vaz et al. "Blood-
retinal
barrier",
Eur J Ophthalmo1-2011, Vol. No.21(S6), pp.:3-9). These retinal diseases may
be due to an impairment of the of the external blood-retinal barrier thus
resulting in retinal edema. Indeed, the dysfunction (alteration or impairment)
of the external blood-retinal barrier leads to an increase of the permeability
of
this barrier. In a preferred embodiment, the retinal disease cursing with
dysfunction of the external blood-retinal barrier, or which is the same, the
retinal disease due to an impairment of the external blood-retinal barrier
thus
resulting in retinal edema, is selected from the group consisting of age-
related macular degeneration, macular edema, retinitis pigmentosa, and
diabetic retinopathy. All these diseases share as common feature, that the
external blood-retinal barrier, namely the layer constituted by the RPE, is
disrupted for several causes. This pathological condition leads to
abnormalities in the vision, which is perceived as dark-spotted, not clear,
impaired or there is no vision.
Thus, in a particular embodiment, the antibody or a fragment thereof that
specifically binds to hemopexin according to the invention is for use in the
treatment and/or prevention of a retinal disease selected from the group
consisting of age-related macular degeneration, macular edema, retinitis
pigmentosa, and diabetic retinopathy.
Therefore, the invention relates to an antibody or a fragment thereof that
specifically binds to hemopexin for use in the treatment and/or prevention of
retinal diseases.
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Macular edema occurs when fluid and protein deposits collect on or under the
macula of the eye (a yellow central area of the retina) and causes it to
thicken
and swell. This is generally due to the disruption of the BRB. The swelling
may distort a person's central vision, as the macula is near the center of the
retina at the back of the eyeball. This area holds tightly packed cones that
provide sharp, clear central vision to enable a person to see detail, form,
and
color that is directly in the direction of gaze. Macular edema is classified
in
cystoid macular edema (CME) or difuse macular edema.
Diabetic retinopathy (DR) remains the leading cause of blindness among
working-age individuals in developed countries. Whereas proliferative
diabetic retinopathy (PDR) is the commonest sight-threatening lesion in type
1 diabetes, diabetic macular edema (DME) is the primary cause of poor visual
acuity in type 2 diabetes. Because of the high prevalence of type 2 diabetes,
DME is the main cause of visual impairment in diabetic patients. In a large
population-based study, the incidence of DME over a period of 10 years was
% in patients with type 1 diabetes whereas this rate was almost 40 % in
patients with type 2 diabetes (Tong et al., "Association of macular
20 involvement with proliferative retinopathy in Type 2 diabetes", Diabet
Med-
2001, Vol. No.18, pp.:388-94). In addition, DME is almost invariably present
when PDR is detected in type 2 diabetic patients. Neovascularization due to
severe hypoxia is the hallmark of PDR whereas vascular leakage due to the
breakdown of the BRB is the main event involved in the pathogenesis of
DME.
Age-related macular degeneration (AMD) is a medical condition which usually
affects older adults and results in a loss of vision in the center of the
visual
field (the macula) because of damage to the retina. Macular degeneration can
make it difficult or impossible to read or recognize faces, although enough
peripheral vision remains to allow other activities of daily life. The leading
cause of visual loss among elderly persons is macular degeneration, signs of
which use to appear after the age of 50. As documented in the western world
it is a leading cause of permanent visual loss with a prevalence of 8.5% in
persons under 54 years of age and of 37% in persons over 75 years of age.
AMD occurs with degeneration of the macula, which is the part of the retina
responsible for the sharp, central vision needed to read or to drive. It is
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diagnosed as either dry (non-neovascular) or wet (neovascular). Neovascular
refers to growth of new blood vessels in the macula, where they are not
supposed to be. The dry form is more common than the wet one, with about
85-90 percent of the patients diagnosed. The wet form of the disease usually
leads to more serious vision loss.
Retinitis pigmentosa (RP) designates a group of inherited diseases that affect
the retina and are characterized by a gradual destruction of the rods and
cones, resulting in a progressive loss of vision and, possibly, blindness.
Usually, the rod cells are the first to degenerate, causing night blindness
and
'tunnel vision. Loss of central vision late in the course of the disease may
occur in some cases. The rate of progression varies. To date, there is no
known way to halt the degeneration of the retina or to cure the disease.
The antibody or fragment thereof, referred in the aspects of the invention,
are
useful in the treatment of all these diseases because they can impede or
minimize the alteration of the retinal pigment epithelium forming part of the
external blood-retinal barrier.
In a preferred embodiment, the antibody or fragment thereof, referred in the
aspects of the invention, specifically binds to mammal hemopexin, more
preferably to human hemopexin. In a preferred embodiment, the antibody or
fragment thereof binds to human hemopexin of SEQ ID NO: 1. In a preferred
embodiment, the antibody or fragment thereof, specifically binds to SEQ ID
NO: 2, which is an epitope of human hemopexin defined by amino acids 50 to
100 of SEQ ID NO: 1. In another preferred embodiment, the antibody or
fragment thereof, referred in the first and second aspects, specifically binds
to
SEQ ID NO: 3, which is an epitope of human hemopexin defined by amino
acids 163 to 462 of SEQ ID NO: 1.
In yet another preferred embodiment, the antibody or fragment thereof is a
polyclonal antibody.
Also in another preferred embodiment, the antibody or fragment thereof is a
monoclonal antibody.
The antibody or a fragment thereof, which specifically binds to hemopexin, for
9
use as defined above, may be a part or an ingredient of a pharmaceutical
and/or
veterinary composition.
In a preferred embodiment, the antibody or a fragment thereof, which
specifically
binds to hemopexin, for use as defined above, is part or an ingredient of a
topical
pharmaceutical and/or veterinary composition. Most preferably, the antibody or
a
fragment thereof is part or an ingredient of a topical composition for ocular
administration, such as a liquid preparation (eye drops), or an ointment.
Alternatively, the antibody or a fragment thereof, which specifically binds to
hemopexin, for use as defined above, may be part or an ingredient of an
injectable
solution or suspension, preferably an intravitreal injectable liquid
(suspension or
solution). Also alternatively, the antibody or fragment thereof may be part of
an orally
administrable pharmaceutical and/or veterinary composition in form of tablets,
pills,
capsules, microcapsules, granules, suspensions, syrups, freeze-dried powders,
liquid preparations, etc. Selection of the excipients and the most appropriate
methods for formulation in view of the particular purpose of the composition
(topical,
injectable or oral administration) is within the scope of ordinary persons
skilled in the
art of pharmaceutical technology.
For example, when eye drops have to be formulated, they must be isotonic with
drops and the solutions, suspensions or ointments include concentrations of
salts
from 0.7 % to 0.9 % w/w, for example of sodium chloride or disodium phosphate
as
buffering agents, preservatives, such as polyvinyl alcohol, and viscosity
agents to
assure permanence in the eye. Examples of viscosity agents include, among
others
methylcellulose derivatives (i.e. methylcellulose). On the other hand, when
the
antibodies or fragments thereof form part of injectable compositions, for
example for
intravitreal injections, they include water for injectables, TweenTm 20,
trehalose
trihydrate and buffering agents, such as disodium phosphate or salts of
aminoacids
(for example histidine salts).
Another aspect of the invention includes a pharmaceutical and/or veterinary
composition that comprises more than one antibody or a fragment thereof that
specifically binds to hemopexin. The pharmaceutical and/or veterinary
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composition preferably comprises more than one antibody or fragment thereof
that specifically binds to hemopexin, said antibodies or fragments thereof
specifically binding different epitopes of hemopexin, or the same epitope with
different sensibilities and specificities. This can also be formulated as a
5 pharmaceutical and/or veterinary composition comprising an effective
amount
of an antibody or a fragment thereof that specifically binds to hemopexin for
use in the treatment and/or prevention of a retinal disease, in particular a
retinal disease cursing with dysfunction of the external blood-retinal
barrier,
together with any pharmaceutically acceptable excipient and/or carrier.
In a preferred embodiment, the invention provides a pharmaceutical and/or
veterinary composition that contains at least an effective amount of an
antibody or a fragment thereof that specifically binds to human hemopexin of
SEQ ID NO: 1, for use in the treatment and/or prevention of a retinal disease,
in particular, a retinal disease cursing with dysfunction of the external
blood-
retinal barrier, together with adequate amounts of pharmaceutically or
veterinary acceptable excipients.
In a most preferred embodiment, the pharmaceutical and/or veterinary
composition comprises an antibody or fragment thereof that specifically binds
to SEQ ID NO: 2, and/ or an antibody or fragment thereof that specifically
binds to SEQ ID NO: 3, and/or any other antibody or a fragment thereof that
specifically binds to any epitope of human hemopexin of SEQ ID NO: 1.
SEQ ID NO: 2 corresponds to the of human hemopexin defined by amino
acids 50 to 100 of SEQ ID NO: 1.
SEQ ID NO: 3 corresponds to the epitope of human hemopexin defined by
amino acids 163 to 462 of SEQ ID NO: 1.
Also preferred is a pharmaceutical and/or veterinary composition consisting in
an antibody or fragment thereof that specifically binds to SEQ ID NO: 2, and
adequate amounts of pharmaceutically or veterinary acceptable excipients.
Another preferred pharmaceutical and/or veterinary composition consists in
an antibody or fragment thereof that specifically binds to SEQ ID NO: 3, and
adequate amounts of pharmaceutically or veterinary acceptable excipients.
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Also another preferred embodiment is a pharmaceutical and/or veterinary
composition that consists in an antibody or fragment thereof that specifically
binds to SEQ ID NO: 2, an antibody or fragment thereof that specifically binds
to SEQ ID NO: 3, and adequate amounts of pharmaceutically or veterinary
acceptable excipients. Another preferred embodiment is a pharmaceutical
and/or veterinary composition that consists in an antibody or fragment thereof
that specifically binds to SEQ ID NO: 2, an antibody or fragment thereof that
specifically binds to SEQ ID NO: 3, an antibody or a fragment thereof that
specifically binds to a sequence of human hemopexin of SEQ ID NO: 1, said
sequence being different from SEQ ID NO: 2 and SEQ ID NO: 3, and
adequate amounts of pharmaceutically or veterinary acceptable excipients.
In this regard, the pharmaceutical and/or veterinary composition for the use
according to the invention may be prepared to be administered by several
means, especially including topical administration, most preferably ocular
topical administration in the form of liquid preparations (solutions,
suspensions) to be applied as eye drops, or in the form of ointments or
creams also applicable to the eyes.
Thus, in a preferred embodiment of the invention, the pharmaceutical and
veterinary compositions are topical compositions. In a more preferred
embodiment, the topical composition is a topical composition for ocular
administration, such as a liquid preparation (eye drops), or an ointment.
Although topical administration is preferred, other forms are possible, such
as
injectable or oral administration. Therefore, the composition containing the
effective amount of the antibody/ies or fragment/s thereof can be
administered as an injectable solution or suspension, preferably an
intravitreal injectable liquid (suspension or solution). Also alternatively,
the
composition can be administered orally in form of tablets, pills, capsules,
microcapsules, granules, suspensions, syrups, freeze-dried powders, liquid
preparations, etc. Selection of the excipients and the most appropriate
methods for formulation in view of the particular purpose of the composition
(topical, injectable or oral administration) is within the scope of ordinary
persons skilled in the art of pharmaceutical technology.
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The term "pharmaceutically acceptable" as used herein pertains to
compounds, materials, compositions, and/or dosage forms which are, within
the scope of sound medical judgment, suitable for use in contact with the
tissues of a subject (e.g. human) without excessive toxicity, irritation,
allergic
response, or other problem or complication, commensurate with a reasonable
benefit/risk ratio. Each carrier, excipient, etc. must also be "acceptable" in
the
sense of being compatible with the other ingredients of the formulation.
Suitable carriers, excipients, etc. can be found in standard pharmaceutical
texts, and include, as a way of example preservatives, agglutinants,
humectants, emollients, and antioxidants. Likewise, the term "veterinary
acceptable" means suitable for use in contact with the tissues of a non-human
animal.
The term "effective amount" as used herein, means an amount of an active
agent (antibody or fragment thereof) high enough to deliver the desired
benefit (either the treatment or prevention of the illness), but low enough to
avoid serious side effects within the scope of medical judgment.
Throughout the description and claims the word "comprise" and variations of
the word, are not intended to exclude other technical features, additives,
components, or steps. Furthermore, the word "comprise" and its variations
encompasses the term "consisting of". Additional objects, advantages and
features of the invention will become apparent to those skilled in the art
upon
examination of the description or may be learned by practice of the invention.
The following examples and drawings are provided by way of illustration, and
they are not intended to be limiting of the present invention. Furthermore,
the
present invention covers all possible combinations of particular and preferred
embodiments described herein.
EXAMPLES
Example 1. Hemopexin disrupts external blood-retinal barrier.
In order to illustrate the effect of the antibodies or fragments thereof for
use in
the treatment and/or prevention of a retinal disease due to an impairment of
(or cursing with dysfunction of) the external blood-retinal barrier, namely
with
alteration of RPE, the inventors developed a permeability assay with the cell
13
line ARPE-19 (AT, Manassas, VA), which is an spontaneous immortalized cell
line of RPE. Cultures were maintained at 37 C and CO2 (5%) in bottles of 75
cm2
and standard media (DMEN Ham's F-12) supplemented with fetal bovine serum at
% (SBF, Hyclone, Cultek, Barcelona, Spain). Streptomycin (100 mg/ml) and
5 penicillin (100 Wm!) were added as preservatives. Glucose concentration
was
adjusted at 25 mM. The media was replaced every 3 days. ARPE-19 cells from
passage 20 were used for permeability studies.
The permeability of the external BRB which is the one including the RPE was
10 analyzed following the methodology disclosed by Garcia-Ramirez M et al.,
"Measuring Permeability in Human Retinal Epithelial Cells (ARPE-19):
implications for the Study of Diabetic Retinopathy", Methods Mol Biol- 201 1 ;
Vol.
No. 763, pp.:179-94
Briefly, ARPE-19 cells were seeded at a density of 400.000 cells/ml that
represented
80.000 RPE cells/well in polystyrene inserts having a surface of 0.33 cm2 (HTS-
TranswellsTm; Costar; Corning Inc, NY, USA). At this density the cells formed
a
monolayer, which was cultivated for 15 days, replacing the media every 3 days.
At
day 15 different treatments (4 replicates/ treatment) were applied via the
apical part
of the wells:
Treatment with hemopexin
Apical media of the insert in the apical part was replaced by deprived serum
media
(Bovine fetal serum, BFS at 1 %), and plasmatic hemopexin was added (50 pg mL,
SIGMA, Madrid, Espana). 15 hours later, fluorescent dextrane was added (10
kDa;
SIGMA, Madrid, Espana) at 100 pg/mL. Afterwards 200 pL of media in the basal
part
of the insert were removed at intervals of 30 min, and replaced by fresh
media.
Absorbancies were read at an exciting wavelength of 485 nm, and an emission
wavelength of 528 nm in the spectrophotometer SpectraMax GeminiTm (Molecular
Devices, Sunnyvale, CA). Dextran concentration was determined by fluorescence
extrapolation in a standard curve.
Treatment with antibodies anti-human hemopexin of SEQ ID NO: 1.
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For the treatment with antibodies, media with hemopexin (50 pg/nnL) was
prepared in
the tubs and the extract concentration of antibody required (0.75 pg/mL) was
added
to the solution. Then, the solutions were vortexed briefly and incubated 1 h.
at 37 C.
The antibodies were: goat anti-human hemopexin antibody Ab D17 (treatment HPX+
Ab D17) (Santa Cruz Biotechnology, Inc, Santa Cruz, CA, USA); rabbit anti-
human
hemopexin antibody Ab H-300 (treatment HPX+ Ab H-300) (Santa Cruz
Biotechnology,
Inc, Santa Cruz, CA, USA); and mouse anti-human hemopexin Ab 013 (treatment
HPX+ Ab 013) (Santa Cruz Biotechnology, Inc, Santa Cruz, CA, USA).
Goat anti-human hemopexin antibody Ab D17 is a polyclonal antibody that
specifically binds to SEQ ID NO: 2, which is the epitope of human hemopexin
defined by amino acids 50 to 100 of SEQ ID NO: 1.
Rabbit anti-human hemopexin antibody Ab H-300 is a polyclonal antibody that
specifically binds to SEQ ID NO: 3, which is the epitope of human hemopexin
defined by amino acids 163 to 462 of SEQ ID NO: 1.
Mouse anti-human hemopexin Ab 013 is a monoclonal antibody raised against full
lenght native hemopexin of human origin.
FIG. 2 shows the dextrane permeability of ARPE-19 cells, measured as the
detectable
fluorescent dextrane in the 200 pL of media removed from the basal part of the
inserts (at 3 minutes from addition of the treatment or at 180 minutes), of
the
treatment with hemopexin (HPX) and antibodies. The treatment with hemopexin
produces a meaningful permeability increase, as deduced from the high bar HPX,
said
increase being prevented if hemopexin was neutralized with the different
antibodies.
On the other hand, an imnnunohistochemical analysis of the ARPE-19 monolayer
was done. Cells that were submitted to the action of HPX, or HPX and one of
the
antibodies mentioned above.
For the treatment with antibodies a solution of 50 pg/mL of hemopexin was
prepared
in EppendorfTM in deprived media (DMEN Ham's F-12 SBF 1 %).
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Further, a solution with the antibody was also added. The solution was
vortexed and
incubated for 1 h at 37 C.
For this assay ARPE-19 cell monolayers grown on crystal for 15 days (Thermo
scientific,
Menzel-Glaser; Braunschweig, GE), were treated with hemopexin and the
antibodies
and then were fixed with cool methanol (-20 C) for 10 minutes, washed with
phosphate
buffer saline (PBS), and blocked and permeabilized overnight (at 4 C) with
bovine serum
albumine (at 2%) in PBS and TweenTm 0.05 %. Further, they were incubated with
primary antibody mouse anti-human ZO-1 (zone occludens-1 ; 1 :200, Zymed
Laboratories
Inc., San Francisco, CA, ) 1 h. As secondary antibody Alexa 594 anti-mouse (1
:200,
Invitrogen, San Diego, CA) was used for 1 h.
Preparations were mounted with a fluorescence mounting medium containing
4',6-diamidino-2-phenylindole (DAPI) to stain nuclei (Vector Laboratories;
Burlingame, CA), and then were visualized in a spectral confocal microscope
FV1000TM (Olympus, Hamburg, Germany). Originally images were captured in the
immersion microscope objective lense (at 60X).
The result of this assay can be seen in FIG. 1. The control (C, no treatment)
shows
organized cells, meanwhile in the treatment with hemopexin (HPX) a
disorganized
monolayer can be seen. Prevention of disorganization is achieved with
antibodies
anti-human hemopexin.
All these data taken together allow concluding that, surprisingly, the
antibodies raised
against hemopexin avoid the disruption of a model of retinal pigment
epithelium, thus
being able to treat retinal diseases, particularly retinal diseases cursing
with alteration of
retinal pigment epithelium, and more generally cursing with dysfunction of the
external
blood-retinal barrier, being the dysfunction an alteration or impairment of
the blood-
retinal barrier for any etiology or cause.
Besides, and with the aim of investigating more in-depth the mechanisms of
action involved
in this process, the inventors have provided herewith evidences of the role of
hemopexin as
an agent able to disrupt the RPE, thus causing the disorganization if
intercellular unions
(tight junctions). This disorganization of one of the main component of the
external blood-retinal
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16
barrier functionally affects the entire barrier, for example, causing an
increased permeability. Increasing the permeability of the barrier leads to
serious problems derived from altered control of the transport of liquid and
soluble compounds through the blood-retinal barrier, such as nutrients. In
addition the impairment of the barrier makes the retina accessible to toxic
compounds.
The embodiments proposed in the present invention alone or taken in
combination with each other, as well as with the examples above disclosed,
allow concluding that antibodies or fragments thereof which can specifically
bind to several hemopexin epitopes, are able to efficiently block the
interaction of hemopexin with its receptor sited in the retina, thus
preventing
the disruption of the RPE, or allowing the restitution of the disrupted RPE.
This represents even an interesting and also efficient approach for treating
and/or preventing a retinal disease, in particular those cursing with
dysfunction of the external blood-retinal barrier, among which there are
included diseases evolving high functional limitations due to the effect of
compromising vision.
REFERENCES CITED IN THE APPLICATION
- Cunha-Vaz et al., "The Blood¨Retinal Barrier in Retinal Disease",
European Ophthalmic Review -2009, Vol. No. 3, pp.:105-108
- Ramirez et al., "Proteomic Analysis of Human Vitreous Fluid by DIGE:
a New Strategy for Identifying Potential Candidates in the
Pathogenesis of Proliferative Diabetic Retinopathy", Diabetologia
2007, Vol. 50, pp.:1294-1303.
- Gao et al., "Characterization of Vitreous Proteome in Diabetes without
Diabetic Retinopathy and Diabetes with Proliferative Diabetic
Retinopahty", Journal of Proteome Research - 2008, vol. 7, pp. 2516-
2525.
- Hernandez et al. "New pathogenic candidates for diabetic macular
edema detected by proteomic analysis", Diabetes Care 2010; 33:e92.
- Garcia-Ramirez M et al., "Measuring Permeability in Human Retinal
Epithelial Cells (ARPE-19). Implications for the Study of Diabetic
Retinopathy". Methods Mol Biol- 2011; Vol. No. 763, pp.:179-94.
- Cunha-Vaz et al. "Blood-retinal barrier", Eur J Ophthalmol- 2011, Vol.
CA 02871170 2014-10-21
WO 2013/164290
PCT/EP2013/058836
17
No. 21(S6), pp.:3-9.
- Tong et al., "Association of macular involvement with proliferative
retinopathy in Type 2 diabetes". Diabet Med-2001, Vol. No. 18,
pp. :388-94.
- WO 2008087049
- KR 100792630
- W02008141285
- JP 2002220349
